KR101080587B1 - Novel Tissue Factor Targeted Antibodies as Anticoagulants - Google Patents

Abstract

The present invention binds to factor VIIa / tissue factor (FVIIa / TF) complexes with greater affinity than tissue factor (TF) alone, does not compete with FVII and FX in binding to TF, and inhibits FX activation. It relates to an antibody. Antibodies of the invention bind to the site of injury and prevent the onset of thrombosis. Antibodies of the invention can be used to treat a variety of thrombotic symptoms, including but not limited to deep vein thrombosis, disseminated intravascular coagulation and acute heart syndrome.

Thrombosis, factor VIIa / tissue factor (FVIIa / TF) complex, FX activation.

Description

Novel Tissue Factor Targeted Antibodies as Anticoagulants

Proper balance between procoagulant activity and anticoagulant activity in blood vessels is essential for normal hemostasis (Davie, E.W. et al. (1991) Biochemistry, 30 (43): 10363-10370). Unbalanced coagulation causes thrombosis, which can lead to heart attack, seizures, pulmonary embolism and venous thrombosis. There is a need for more effective and safe anticoagulants to treat certain thrombotic diseases.

Tissue factor (“TF”) is a transmembrane glycoprotein that is the main initiator of the staged coagulation reaction (Nemerson, Y. (1995) Thromb. Haemost. 74 (1): 180-184). Under normal physiological conditions, active TF is not in contact with blood. Upon vascular injury, the exposure of endothelial TF and collagen to the blood activates coagulation factors and platelets to form hemostatic plugs. Exposed TF acts as a cofactor for catalytic activation of Factor IX ("FIX") and Factor X ("FX"), which are key components of the endogenous tenase and prothrombinase complexes, respectively do. This results in the rapid formation of FXa and thrombin. Thrombin then cleaves the fibrinogen into fibrin, which then fibrin polymerizes to form fibrin blood clots. In various clinical conditions, improperly induced expression of TF can result in life-threatening thrombosis and / or pathological complications. TF exposure accompanying plaque destruction is believed to be responsible for thrombus obstruction resulting in acute myocardial infarction and seizures. In this condition, the proinflammatory signaling pathway activated by the coagulation factor also results in edema formation and increases the infarct site. Vascular damage associated with angioplasty upregulates TF on SMCs believed to induce cellular signaling pathways associated with restenosis. TF overexpression in cancer and gram-negative sepsis results in the activation of life-threatening thrombosis and inflammatory pathways.

FVIIa / TF complexes are involved in the pathogenic mechanism of various thrombotic diseases, and in some patients a circulating amount of TF is a risk factor. FVIIa and TF play an important role in maintaining hemostasis and initiating thrombus formation in vascular injury. TF is normally expressed in the outer membrane, but in vascular disease it is inappropriately upregulated and expressed in the middle and neovascular lining of blood vessels. In atherosclerosis plaques, TF expression increases and is protected from blood by a thin fibrous cap that, when broken, exposes TF. Surgical procedures, such as balloon angioplasty, stenosis, or endometrial resection, damage the vessel wall and expose the TF below it. In atherosclerosis, spontaneous destruction or endothelial erosion of lipid-rich thin-walled plaques results in TF exposure and thrombus formation leading to unstable angina and myocardial infarction. TF can be circulated in microparticles derived from cells and circulating TF levels increase in unstable angina, suggesting that circulating TF can lead to thrombus formation (Soejima, H. et. al. (1999) Circulation 99 (22): 2908-2913]. Cancer is largely associated with an overcoagulation state due to TF overexpression on tumor cells. This makes patients susceptible to deep vein thrombosis, pulmonary embolism and low grade disseminated intravascular coagulation (“DIC”). DIC causes fibrin deposition that results in multiple organ failure in microvessels.

Protein-based anticoagulants targeting TF include TF neutralizing antibodies, active site inhibited factor VIIa ("FVIIai"), tissue factor pathway inhibitors ("TFPI"), and nematode anticoagulant proteins ("NAPC2"). . Results from the acute arterial injury model of thrombosis suggest that protein-based inhibitors of FVIIa / TF are effective antithrombotic agents and have less bleeding than heparin, direct thrombin inhibitors, platelet inhibitors, and FXa inhibitors [Himber, J. et al. (2001) Thromb. Haemost. 85: 475-481; Harker, L. A. et al. (1995) Thromb. Haemost. 74 (1): 464-472]. In addition, FVIIa / TF inhibition is superior to other anticoagulants (eg, heparin, FXa inhibitors) in preventing the thickening and vascular narrowing of the neovascular endothelial involved in balloon injury (Jang, Y. et al. (1995) Circulation 92 (10): 3041-3050].

Inhibiting TF, FVIIa or FVIIa / TF complexes is an effective antithrombotic method of preventing DIC and reducing mortality in experimental models of sepsis. TFPI analogs prevent both thromboplastin and endotoxin-induced DIC in rabbits [Day, KC et al. (1990) Blood 76: 1538-1545; Bregengard, C. et al. (1993) Blood Coagul. Fibrinolysis 4: 699-706]. Monoclonal antibodies against FVIIa (Biemond, BJ et al. (1995) Thromb. Haemost. 73: 223-230 or Levi, M. et al. (1994) J. Clin. Invest. 93: 114-120) Prevent endotoxin-induced DIC in monkeys. TF neutralizing antibodies, FVIIai and TFPI. Inhibits DIC and reduces mortality in a baboon model of E. coli -induced sepsis [Creasey, AA et al. (1993) J. Clin. Invest. 91: 2850-2860; Taylor, FB et al. (1991a) Blood 78: 364-368; Taylor, FB et al. (1991b) Circ. Shock 33: 127-134; Taylor, FB (1996) Haemostasis Suppl. 126: 83-91. Both free FXa and FVIIa / TF / FXa complexes are known to induce the production of proinflammatory cytokines associated with an increased risk of death in patients with sepsis [Riewald, M. et al. (2001) Proc. Natl. Acad. Sci. USA 98: 7742-7747. Interestingly, FVIIai has been shown to lower plasma levels of IL-6 and IL-8 in the baboon model [Taylor, FB et al. (1998) Blood 91: 1609-1615], suggesting that inhibition of FVIIa / TF may have additional anti-inflammatory effects that are not shared by other anticoagulant mechanisms.

Several antibodies have been described that are effective anticoagulants and bind to and neutralize TF or FVIIa / TF complexes or both (see, eg, Carson, SD et al. (1985) Blood 66 (1): 152-156; Tanaka, H. et al. (1985) Thromb. Res. 40 (6): 745-756; Kirchhofer, D. et al. (2000) Throomb. Haemost. 84 (6); 1072-1081; Kirchhofer, D. et al. (2001) Biochemistry 40 (3): 675-682; Faelber, K. et al. (2001) J. Mol. Biol. 313: 83-97; and US Pat. Nos. 5,506,134, 5,986,065 And 6,274,142). The TF targeted antibodies of the present invention are effective anticoagulants with improved characteristics compared to the TF antibodies described earlier in the present invention. In particular, the antibodies of the invention bind with a greater affinity for the FVIIa / TF complex than TF alone, and do not compete with FVII or FX for binding to TF.

<Overview of invention>

The present invention provides antibodies that act as anticoagulants that bind with greater affinity for the Factor VIIa / Tissue Factor (“FVIIa / TF”) complex than the Tissue Factor (“TF”) alone. In one embodiment, the antibodies of the invention bind with affinity at least two times greater for the FVIIa / TF complex than TF alone as measured in the microcalorimetry assay. In a preferred embodiment, the antibodies of the invention bind with at least five times greater affinity for the FVIIa / TF complex than the TF alone. In a more preferred embodiment, the antibodies of the invention bind with at least 10 times greater affinity for the FVIIa / TF complex than the TF alone. In another embodiment, the antibody of the invention is at least one coagulation factor selected from the group consisting of Factor VII ("FVII"), Factor IX ("FIX") and Factor X ("FX") in binding to TF. Do not compete with In a preferred embodiment, the antibodies of the invention do not compete with FVII and FX in binding to TF. In a more preferred embodiment, the antibodies of the invention bind with greater affinity with the FVIIa / TF complex than with TF alone, and do not compete with FVII and FX for binding to TF.

Anticoagulant antibodies of the present invention prevent thrombus formation by targeting the FVIIa / TF complex at the site of injury and binding to the complex to inhibit factor X ("FX") activation, thereby preventing sepsis, disseminated intravascular coagulation, It effectively acts as an anticoagulant in the treatment of several diseases including ischemic attacks, deep vein thrombosis, acute heart syndrome, thrombotic complications following angioplasty, and coagulation in advanced cancers. The antibodies are also used in microvascular surgery, skin and vein transplantation, and organ transplantation.

In another aspect, the present invention provides a pharmaceutical composition comprising the antibody of interest.

In another aspect, the invention comprises inhibiting thrombin production by administering to a patient to be protected from thrombus formation a therapeutically effective amount of the antibody of the invention without directly affecting other coagulation parameters such as activation and aggregation of platelets. And providing a method for protecting said patient from thrombus formation.

In another aspect, the invention provides a method of administering a therapeutically effective amount of an antibody of the invention to a patient suffering from deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), acute heart syndrome, or cancer with signs of coagulation. A method of alleviating and treating deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), acute heart syndrome, or cancer with signs of coagulation in said patient.

In another aspect, the invention relates to a method of modulating an inflammatory response in a patient comprising administering a therapeutically effective amount of the antibody of the invention to a patient in need thereof.

In another aspect, the antibodies of the invention can be used to form a coating that prevents thrombus formation on the surface of a medical device in contact with blood.

In another aspect, the invention relates to a kit comprising an antibody of the invention which binds to a FVIIa / TF complex. Alternatively, the kit may comprise a DNA sequence encoding an antibody component.

Also disclosed are methods for producing antibodies of the invention by recombination and synthesis.

1 shows the activity of TF-binding single chain antibodies in sTF / FVIIa peptide hydrolysis assay. sTF / FVIIa peptide hydrolysis assay using Example 4 equilibrium mixture of FVIIa (5 nM) and sTF (10 nM) based on the affinity of FVIIa for sTF (K _{D (app)} is about 10 nM) It was performed as described in. Hydrolysis of the chromogenic peptide substrate S2266 was monitored as described. Final concentrations of the single chain antibody and control protein FVIIai (FVIIa inactivated by PPACK, a chloromethyl ketone peptide) and Mab # 4504 (American Diagnostica) expressed by bacteria are shown.

2 shows that binding of scFv (TF) 3e10 with sTF increases the affinity of sTF for FVIIa. sTF / FVIIa peptide hydrolysis was performed as described in Example 4 using 2 nM FVIIa in the presence and absence of 800 nM scFv (TF) 3e10 expressed by bacteria. The sTF was titrated in the assay and the cleavage rate of the chromogenic peptide substrate S2266 was measured. The K _D (apparent) for sTF was calculated using a standard 4-parameter fit.

3 shows the activity of TF-binding single chain antibodies in prothrombin time (PT) analysis. PT analysis was performed as described in Example 4 using recombinant human thromboplastin (Dade, Inc.) containing full length human TF in phospholipid vesicles. Final concentrations of the single chain antibody and control protein FVIIai expressed by the bacteria are shown.

4 shows the measurement of the apparent binding affinity of scFv (TF) 3e10 for sTF. sTF / FVIIa peptide hydrolysis analysis was performed as described in Example 4 using 3 nM sTF and 2 nM FVIIa. The concentration of sTF used was less than K _D in binding to FVIIa. Was added while the scFv (TF) 3e10 expressed by the bacteria and increase the concentration, was used to increase the reaction rate determining K _D (apparent) of the antibody for sTF, it was used as a standard 4-parameter feet. The affinity of scFv (TF) 3e10 for the sTF / FVIIa complex (K _{D (app)} = 65 nM) is the affinity of scFv (TF) 3e10 for sTF measured using BIAcore (K _{D (app)} = 470 nM).

5 shows the determination of the binding affinity of scFv (TF) 3e10 for TF and FVIIa / TF complexes. Microcalorimetry analysis was performed as described in Example 4 using scFv (TF) 3e10 expressed in CHO cells. This analysis shows that scFv (TF) 3e10 has about 20 times greater affinity for the sTF / FVIIa complex (“complex”) than sTF (“free TF”).

6 shows that scFv (TF) 3e10 inhibits FX activation in a dose dependent manner. FX activation assay was performed as described in Example 4 with increasing concentrations of scFv (TF) 3e10, 250 nM FX, and FVIIa / TF complex (10 pM FVIIa) expressed on the phospholipid surface. IC ₅₀ represents the dosage required to reach 50% maximal inhibition.

7 shows that scFv (TF) 3e10 inhibits FX activation non-competitively by the FVIIa / TF complex. FX activation assay was performed as described in Example 4 using scFv (TF) 3e10 expressed by bacteria, 25 nM to 400 nM FX, and FVIIa / TF complex (10 pM FVIIa) on phospholipid surfaces. The assay was titrated with increasing concentration of scFv (TF) 3e10 (0 nM, hollow square; 0.25 nM, hollow diamond; 0.74 nM, hollow triangle, 2.2 nM, hollow circle; 6.7 nM, filled diamond) Type, 20 nM, filled triangle). (1 / [S], where S (substrate) = factor X (μM); vs 1 / v, where v (rate) = mOD / min (obtained from S2222 hydrolysis with FXa generated over 5 minute intervals)) The Lineweaver-Burk graph of shows that scFv (TF) 3e10 is an uncompetitive inhibitor for substrate FX. All lines cross the x-axis (or near it) as expected for noncompetitive inhibitors (for competitive inhibitors, all lines follow the y-axis).

8 shows that scFv (TF) 3e10 is efficacious in an in vivo model of disseminated intravascular coagulation (“DIC”). TF-antibody scFV (TF) 3e10 expressed in CHO cells was evaluated in the rat thromboembolism model described in Example 6 for (A) mortality rate and (B) morbidity-mortality score. (A) In the vehicle-treated group, the dose of TF used exhibited a mortality of 60% (LD ₆₀ ). 0.7 nmol / kg of scFv (TF) 3e10 did not affect mortality, while 7.0 nmol / kg of scFv (TF) 3e10 reduced mortality to less than 40%. In the vehicle-treated group (B), the in vivo dose of TF showed an average morbidity-mortality score of 2.6. At 0.7 nmol / kg scFv (TF) 3e10 had no effect on mortality and little or no effect on respiratory impairment, the mean morbidity-mortality score at 14 nmol / kg was reduced to about 1.5.

Anticoagulant antibodies of the invention are antibodies that bind with greater affinity for the Factor VIIa / Tissue Factor (“FVIIa / TF”) complex than the Tissue Factor alone (“TF”). Antibodies of the invention bind with affinity at least twice, preferably at least 5 times, more preferably at least 10 times greater than the FVIIa / TF complex than TF alone. Antibodies of the invention do not compete with one or more coagulation factors selected from the group consisting of Factor VII ("FVII"), Factor IX ("FIX") and Factor ("FX") in binding to TF. Preferably, the antibodies of the invention do not compete with FVII and FX for binding to TF.

Justice:

In the description of the present invention, the following terms are defined as follows.

"Recombinant protein or polypeptide" refers to a protein or polypeptide produced by recombinant DNA technology, ie, transformed by an exogenous DNA construct encoding a polypeptide of interest. Proteins or polypeptides expressed in most bacterial cultures will not contain glycans. Proteins or polypeptides expressed in yeast may have a different glycosylation pattern than that expressed in mammalian cells.

A “natural” protein or polypeptide refers to a protein or polypeptide recovered from a naturally occurring source. The term "natural antibody" will include naturally occurring antibodies and fragments thereof.

DNA “coding sequences” are DNA sequences that are transcribed into mRNA and translated into polypeptides in host cells when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by the start codon of the 5 'N-terminal and the translation end codon of the 3' C-terminal. Coding sequences may include prokaryotic sequences, cDNAs from eukaryotic mRNAs, genomic DNA sequences from eukaryotic DNA and synthetic DNA sequences. Transcription termination sequences will typically be located 3 'of the coding sequence.

A "nucleotide sequence" is a heteropolymer of deoxyribonucleotides (adenine, guanine, thymine or cytosine bases). DNA sequences encoding the antibodies of the invention can be assembled from synthetic cDNA-derived DNA fragments and short oligonucleotide linkers to provide synthetic genes that can be expressed in recombinant expression vectors. In discussing the structure of certain double-stranded DNA molecules, the sequences may be described herein according to the conventional practice of providing sequences in the direction of only 5 'to 3' along the non-transcribed strand of the cDNA.

A "recombinant expression vector" is a replicable DNA construct used to amplify or express DNA encoding an antibody of the invention. Expression vectors contain DNA regulatory sequences and coding sequences. DNA regulatory sequences include promoter sequences, ribosomal binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, and enhancers. The recombinant expression system as defined herein will express the antibody upon induction of regulatory elements.

"Transformed host cell" means a cell transformed and transfected with exogenous DNA. Exogenous DNA may or may not be integrated (or covalently) into the chromosomal DNA that makes up the genome of the cell. For example, in prokaryotes and yeasts, exogenous DNA can be maintained on episomal elements such as plasmids or stably integrated into chromosomal DNA. In eukaryotic cells, stably transformed cells refer to cells in which exogenous DNA is integrated into chromosomal replication. This safety is evidenced by the ability of eukaryotic cell lines or clones to produce a population of daughter cells containing exogenous DNA.

The terms “analogue,” “fragment,” “derivative” and “variant”, when referring to an antibody of the invention, include analogs, fragments, of an antibody having substantially the same biological function or activity, as further described below. Derivatives and variants.

"An analog" includes a pro-polypeptide comprising therein an amino acid sequence of an antibody of the invention. The active antibodies of the invention can be cleaved from additional amino acids that compete with the pro-antibody molecule by natural in vivo processes or by methods well known in the art, such as enzymatic or chemical cleavage. For example, the recombinant scFV (TF) 3e10 polypeptide (SEQ ID NO: 1) is expressed as a pro-polypeptide of 282 amino acids and then processed in vivo to release the active polypeptide of mature 264 amino acids.

A “fragment” is a portion of an antibody of the invention that possesses substantially similar functional activity as shown in the in vitro assays described further herein below.

“Derivatives” are all modifications to the antibodies of the invention that substantially preserve the functions disclosed herein and include additional structural and minor functions, eg, PEGylated with long half-lives, as described further below. Antibodies and biotinylated antibodies. Derivatives also include N- or O-linked glycosylated antibodies that can be generated by inserting N- or O-glycosylation sites into antibody sequences by standard recombinant DNA techniques.

"Substantially similar functional activity" and "substantially the same biological function or activity" are about 30% to 100% of the biological activity demonstrated by the polypeptide of interest when the biological activity of each polypeptide is determined by the same method or assay. Each means a degree of biological activity in the range of% or higher. For example, an antibody having functional activity substantially similar to that of Example 1 (SEQ ID NO: 1) can be combined with the FVIIa / TF complex when tested in the sTF / FVIIa peptide hydrolysis and FX activation assay described in Example 4. It is an antibody that demonstrates its ability to neutralize it.

“Similarity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substituents with that of the second polypeptide. Such conservative substitutions include those described above in The Atlas of Protein Sequence and Structure 5 by Dayhoff (1978) and by Argos (1989) EMBO J. 8: 779-785. For example, amino acids belonging to one of the following groups show conservative changes:

Ala, Pro, Gly, Gln, Asn, Ser, Thr;

Cys, Ser, Tyr, Thr;

Val, Ile, Leu, Met, Ala, Phe;

Lys, Arg, His;

Phe, Tyr, Trp, His; And

-Asp, Glu.

As used herein, "antibody" includes circular immunoglobulin ("Ig") molecules, and fragments thereof such as Fab, F (ab ') ₂ and Fv, which are epitopes, eg, of selected target proteins. In combination with soluble TF ("sTF"). Typically, six, eight, ten or twelve or more contiguous amino acids are needed to form an epitope. However, epitopes comprising non-contiguous amino acids may require more amino acids, for example 15, 25 or 50 or more amino acids.

All other technical terms used herein have the same meaning as commonly used by one of ordinary skill in the art to which this invention belongs.

Antibodies of the Invention and Their Generation :

Anticoagulant antibodies of the invention bind with greater affinity for the Factor VIIa / Tissue Factor (“FVIIa / TF”) complex than the Tissue Factor alone (“TF”). In a preferred embodiment, the antibodies of the present invention are at least 2 times, more preferably at least 5 times, even more preferably at least 10 times more relative to the FVIIa / TF complex than TF alone as determined by microcalorimetry analysis. Bind with great affinity. In another preferred embodiment of the invention, the antibody has at least one coagulation factor selected from the group consisting of Factor VII ("FVII"), Factor IX ("FIX") and Factor X ("FX") in binding to TF. Do not compete with In even more preferred embodiments, the antibodies of the invention do not compete with FVII and FX for binding to TF. In the most preferred embodiment, the antibodies of the invention bind with greater affinity for the FVIIa / TF complex than the TF alone and do not compete with FVII and FX for binding to TF.

Generally speaking, antibodies that specifically bind to selected target proteins (eg, FVIIa / TF complexes or TF) are five, ten or twenty times more than the detection signals provided to other proteins when used in immunochemical assays. Provide a high detection signal. Preferably, the antibody that specifically binds to the selected target protein does not detect other proteins in the immunochemical assay and can immunoprecipitate the target protein in solution.

Polyclonal antibodies can be prepared by immunizing mammals such as mice, rats, rabbits, guinea pigs, monkeys, or humans with selected target proteins. If desired, the target protein can be conjugated to a carrier protein such as bovine serum albumin, tyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants may be used to increase the immune response. Such auxiliaries include Freund's auxiliaries, mineral gels (e.g. aluminum hydroxide), and surface active substances (e.g. lysorecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and di Nitrophenol), but is not limited thereto. Among the adjuvants used in humans, BCG ( bacilli Calmette-Guerin ) and Cornybacterium parvum are particularly useful.

Monoclonal antibodies that specifically bind to the selected target protein can be prepared using any technique for preparing antibody molecules by continuous cell lines in culture. Such techniques include hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology [Kohler et al. (1985) Nature 256: 495-497; Kozbor et al. (1985) J. Immunol. Methods 81: 31-42; Cote et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2026-2030; and Cote et al. (1984) Mol. Cell Biol. 62: 109-120, but is not limited to such.

In addition, techniques developed for the production of “chimeric antibodies”, which are “techniques for splicing mouse antibody genes with human antibody genes to obtain molecules with appropriate antigen specificity and biological activity” can be used [Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855; Neuberger et al. (1984) Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454. It is also possible to "humanize" monoclonal and other antibodies to prevent a patient from performing an immune response against a therapeutically used antibody. Such antibodies may be sufficiently similar to the sequence of the human antibody to be used directly or may require modification of some key residues. Sequence differences between rodent antibodies and human sequences can be minimized by site directed mutagenesis of each residue by replacing residues different from those of the human sequence or by grafting the entire complementarity determining regions. Alternatively, humanized antibodies can be prepared using the recombination method as described in GB2188638B. Antibodies that specifically bind to a selected target protein may contain a partially or fully humanized antigen-binding site as disclosed in US Pat. No. 5,565,332.

Alternatively, techniques for the production of single chain antibodies can be modified using methods known in the art to produce single chain antibodies (“scFv”) that specifically bind to the selected target protein. Antibodies of distinct idiotypic composition with related specificities can be generated by chain shuffling from a random combination of Ig libraries [Burton (1991) Proc. Natl. Acad. Sci. USA 88: 11120-11123.

Single-chain antibodies can also be constructed using DNA amplification methods such as PCR using hybridoma cDNA as a template [Thirion et al. (1996) Eur. J. Cancer Prev. 5: 507-511. Single chain antibodies can be monospecific or bispecific and can be bivalent or tetravalent. The construction of tetravalent bispecific single chain antibodies is described, for example, in Coloma and Morrison (1997) Natl. Biotechnol. 15: 159-163. The construction of bivalent bispecific single-chain antibodies is described in the study of Melendar and Voss (1994) J. Biol. Chem. 269: 199-216.

Nucleotide sequences encoding single-chain antibodies are constructed using manual or automated nucleotide synthesis, cloned into expression constructs using standard recombinant DNA methods, and introduced into cells to express coding sequences. Alternatively, single chain antibodies are described, for example, in fibrous phage display technology [Verhaar et al. (1995) Int. J. Cancer 61: 497-501; and Nicholls et al. (1993) J. Immunol. Meth. 165: 81-91.

In addition, antibodies that specifically bind to selected target proteins can be produced by in vivo production in lymphocyte populations or by Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86: 3833-3837; Winter et al. (1991) Nature 349: 293-299, can also be prepared by screening Ig libraries or panels of high specific binding reagents.

DNA encoding the antibodies of the invention can be cloned into cDNA or genomic form by any cloning method known to those skilled in the art. See, eg, Sambrook, J. F. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989).

In the case where the antibody is a monoclonal antibody, if a DNA sequence encoding the Fv region is identified and exhibits specific binding activity when expressed, an antibody comprising such an Fv region can be prepared by methods known to those skilled in the art. Thus, for example, Chaudhary, V. K. et al. (1989) Nature 339 (6223): 394-397; Batra, J. K. et al. (1990) J. Biol. Chem. 265 (25): 15198-15202; Batra, J. K. et al. (1989) Proc. Natl. Acad. Sci. USA 86 (21): 8545-8549; Chaudhary, V. K. et al. (1990) Proc. Natl. Acad. Sci. USA 87 (3): 1066-1070 describe the preparation of various single chain antibody proteins.

In a preferred method, the TF-binding antibodies of the invention are single chain antibodies prepared using phage display libraries. The epitope-binding region of a single chain antibody consists of two variable region domains, one from the heavy chain and the other from the light chain. In the first step to construct a phage display library, PCR cloning of variable genes (V _H (from IgM) V _κ and V _L ) from collected mRNA from human bone marrow, lymph nodes and spleen using a family specific primer set It was. The resulting pCITE-V _H (3.8 × 10 ⁹ members), pZ604-V _κ (1.6 × 10 ⁷ ), and pZ604-V _L (3.2 × 10 ⁷ ) libraries show permanent high diversity of the V gene. The V _H gene is then amplified from the pCITE-V _H library. V _κ and V _L genes are PCR amplified from pZ604-V _κ and pZ604-V _L libraries with opposite J _H and linker sequences at the 5 ′ end. The scFv gene repertoire is then prepared by splicing together the gel purified V _H , V _κ , and V _L containing the PCR product. The scFv gene repertoire was cloned into the phagemid vector pZ603 and the ligation product was electroporated in water-soluble competent TG1. E. coli cells were added to generate a scFv phage display library HuPhabL3 with 5.2 × 10 ⁹ each transformant [Kay, BK et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego CA; Marks, JD et al. (1991) J. Mol Biol. 222 (3): 581-597; Sheets, MD et al. (1998) Proc. Natl. Acad. Sci USA 95 (11): 6157-6162].

TF-binding phages from scFv phage display libraries are selected, amplified and identified using panning techniques well known in the art. Soluble TF is fixed in a plastic tube and skim milk is used to reduce non-specific binding to the plastic. The population of scFv phages is exposed to sTF immobilized in a plastic tube and unbound phages are washed off entirely. TF-binding scFv phage was eluted from the tube and then TG1 in solution. Infect and amplify E. coli cells. This panning method is repeated three times and the resulting TF-binding scFv phage is isolated by transforming TG1 cells. Transformants expressing TF-binding antibodies are identified using standard ELISA using sTF immobilized on plastic in 96-well dishes. The DNA of the single-chain antibody insert of the ELISA-positive transformants is sequenced. Based on DNA sequencing, six specific single chain antibodies, scFv (TF) 2c1, scFv (TF) 2c11, scFv (TF) 2d3, scFv (TF) 2h6, scFv (TF) 3e10 and scFv (TF) 3h2 As identified herein; It was expressed and purified from E. coli and characterized as described in Example 5 below.

Expression and Purification of Antibodies of the Invention:

this. There are several methods for expressing the recombinant antibodies of the invention in vitro, including coli, baculovirus, yeast, mammalian cells or other expression systems. Methods of expressing cloned genes in bacteria are well known. To express high levels of cloned genes in prokaryotic systems, it is essential to construct an expression vector containing at least a strong promoter that directs mRNA transcription termination. Examples of regulatory regions suitable for this purpose include: Promoter and operator regions of the E. coli beta-glucosidase gene, E. coli. E. coli tryptophan biosynthetic pathway, or the left promoter from phage lambda. this. It is useful to include selection markers in DNA vectors that transform E. coli. Examples of such markers include genes that designate resistance to ampicillin, tetracycline or chloramphenicol.

Among the higher eukaryotic cell systems useful for the expression of the antibodies, analogs, fragments, derivatives or variants thereof of the invention, a number of cellular systems are selected. Examples of mammalian cell lines include, but are not limited to, RPMI 7932, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, C127 or MDCK cell lines. Preferred mammalian cell lines are CHL-1. When using CHL-1, hygromycin is included as a eukaryotic selection marker. CHL-1 cells are derived from RPMI 7032 melanoma cells, a readily available human cell line. The CHL-1 cell line was deposited with the ATCC in accordance with the provisions of the Budapest Treaty, and deposited on June 18, 1987, assigned Accession No. #CRL 9446. Cells suitable for use in the present invention are commercially available from ATCC. Exemplary cell lines include Spodoptera frugiperda and Bombyx mori .

E. prokaryotic system E. coli cannot perform post-translational modifications, eg glycosylation. In addition, proteins with complex disulfide patterns are E. coli. Often expressed in E. coli is misfolded. In prokaryotic systems, the expressed protein is present in the cytoplasm in an insoluble form called an inclusion body, found in soluble aliquots after the cells are lysed, or by the addition of an appropriate secretion signal sequence periplasm). If the expressed protein is present in an insoluble inclusion body, solubilization of the inclusion body and subsequent refolding are generally required.

Many prokaryotic expression vectors are known to those skilled in the art and include, for example, commercially available pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pKK233-2 (Clontech, Palo Alto, CA, USA) and pGEM1 (Promega Biotech, Madison, Wi., USA).

Promoters commonly used in recombinant microbial expression systems include beta-lactamase (penicillinase) and lactose promoter systems (Chang, AC et al. (1978) Nature 275 (5681): 617-624; Goeddel, DV et al) (1979) Nature 281 (5732): 544-548), tryptophan (trp) promoter system (Goeddel, DV et al. (1980) Nucl.Acids Res. 8 (18): 4057-4074) and tac promoter (Maniatis) , T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982). Other useful bacterial expression systems include lambda phage pL promoters and clts857 heat-inducible refreshers (Bernard, HU et al. (1979) Gene 5 (1): 59-76; Love, CA et al. (1996) Gene 176 ( 1-2): 49-53). Recombinant antibodies may also be expressed in yeast hosts, such as Saccharomyces cerevisiae . It is common to provide the ability to perform various post-translational modifications. The expressed antibody can be secreted into the culture supernatant in which many other proteins are not present, thereby making purification easier. Yeast vectors for the expression of the antibodies of the invention include several necessary features. Elements of the vector are generally derived from yeast and bacteria and allow for propagation of plasmids in both of them. Bacterial elements include origins of replication and selectable markers. Yeast elements include replication origin sequences (ARS), selectable markers, promoters and transcription terminators.

Suitable promoters in yeast vectors for expression include the TRP1 gene, the ADH1 or ADHII gene, the acid phosphatase (PH03 or PH05) gene, the promoter of the isocytochrome gene, or the promoters involved in the pathway, for example enolase, glycerol. Promoters of aldehyde-3-phosphate dehydrogenase (GADPH), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate kinase, triosphosphate isomerase and phosphoglucose isomerase. Hitzeman, RA et al. (1980) J. Biol. Chem. 255 (24): 12073-12080; Hess, B. et al. (1968) J. Adv. Enzyme Reg. 7: 149-167; and Holland, M. J. and Holland, J. P. (1978) Biochemistry 17 (23): 4900-4907].

Commercially available yeast vectors include pYES2, pPIC9 (Invitrogen, San Diego, Calif.), Yepc-pADH2a, pYcDE-1 (Washington Research, Seattle, WA), pBC102-K22 (ATCC # 67255), and YpGX265GAL4 (ATCC # 67233). Mammalian cell lines include, but are not limited to, COS-7, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, BHK, CHL-1, NSO, and recombinant HEK293 with the present invention. Antibodies can be expressed. Recombinant proteins produced in mammalian cells are typically soluble, glycosylated, and have a definite N-terminus. Mammalian expression vectors include non-transcribed elements such as origins of replication, promoters and enhancers, and 5 'or 3' untranslated sequences such as ribosomal binding sites, polyadenylation sites, acceptor sites, and Splice donors, and transcription termination sequences. Promoters generally used in mammalian expression vectors include, for example, viral promoters such as polyomas, adenoviruses, HTLVs, Simian virus 40 (SV 40), and human cytomegalovirus (CMV). There is.

Depending on the expression system and host selected, homologous recombinant antibodies can be obtained using various combinations of conventional chromatography used for protein purification. Such chromatography includes immunoaffinity chromatography, reverse phase chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography and HPLC. When the expression system secretes antibodies into the culture medium, the protein can be purified directly from the medium. If the antibody is not secreted, the antibody is isolated from the cell lysate. Cell rupture can be performed by any conventional method, including freeze-thaw cycling, sonication, mechanical rupture or the use of cell lysates.

A plasmid construct (Pharmacia) based on pCANTAB5 was used in bacteria to express the single chain antibodies of the invention. For example, the plasmid containing the single chain antibody scFv (TF) 3e10 is pZ612 / 3e10 and encodes the short chain antibody sequence following the e-tag sequence that can be used for protein purification. The amino acid sequence of the scFV (TF) 3e10 antibody corresponds to SEQ ID NO: 1 (Example 1) and the DNA sequence encoding scFv (TF) 3e10 corresponds to SEQ ID NO: 2.

Plasmid constructs based on pTHR525 (see US Pat. No. 5,827,824) were used in mammals to express single chain antibodies of the invention. For example, primers were designed to generate DNA fragments spanning pro-scFv (TF) 3e10 amino acid sequences, including N-terminal signal sequences and C-terminal e-tag sequences, in bacterial expression plasmids. PCR was performed to generate DNA fragments inserted into mammalian expression plasmids containing ampicillin resistance genes and hygromycin and DHFR selection markers. Expression of the single chain antibodies of the invention was induced by the MPSV LTR promoter.

In a preferred embodiment of the invention, CHO DXB11 cells are transfected with a mammalian expression construct. Stable populations were selected using 400 μg / ml hygromycin B in HAMS / F12 medium. The expression level was approximately 500 μg / L. To increase expression levels, populations were selected using 100 nM methotrexate in alpha MEM medium. Approximate expression level of this population was 5 mg / L.

Antibodies of the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passing over a column to which TF is bound. The bound TF-antibody can then be eluted from the column using a buffer containing a high concentration of salt.

Single chain antibodies described herein contain an e-tag sequence at the C-terminus of the protein. Anti-e-tag affinity column was purchased from American / Pharmacia Biotech. The cell culture medium was filtered through a 0.22 μm filter and loaded on a 5 ml e-tag column at a rate of 2 ml / min. The column was washed with 0.2 M phosphate buffer 0.05% NaN ₃ (pH 7.0) and then collected in a tube containing 0.1 volume of 1 M Tris buffer (pH 8.2) to neutralize the elution buffer. Alternatively, the filtered culture medium was loaded onto a Protein A column. In this case, the column was washed with 50 mM citric acid, 300 mM NaCl, pH 6.5 and eluted with the same buffer at pH 3.0. In both cases, the purified sample was then loaded onto a Sephadex 200 column to separate monomers from the dimeric form of the antibody.

Screening for Antibodies of the Invention:

Once the antibodies are generated, expressed and purified, the BIAcore, sTF dependent factor VIIa assay (sTF / FVIIa peptide hydrolysis assay), FX activation assay, and the PT assay described in Example 4 are used to identify the antibodies of the present invention. Antibodies can be further characterized.

In a preferred embodiment of the invention, TF-binding scFv antibodies that bind with greater affinity for the FVIIa / TF complex than TF alone were selected using sTF / FVIIa peptide hydrolysis assay. In this assay, TF-antibodies that bind with greater affinity for the FVIIa / TF complex than TF are expected to increase K _{D (app)} . 2 shows that the single-chain antibody scFv (TF) 3e10 increased K _{D (app)} by about 5 fold. Microcalorimetry analysis is used to determine the affinity of the TF-antibody for the FVIIa / TF complex relative to the TF alone. 5 shows that the single chain antibody scFv (TF) 3e10 has a K _D of 600 nM and 33 nM in binding to the FVIIa / TF complex and the free TF, respectively, which is about 20 for the FVIIa / TF complex compared to the TF alone. Corresponds to a fold greater affinity. Antibodies of the invention have affinity that is at least 2 times, preferably at least 5 times and more preferably at least 10 times greater than the FVIIa / TF complex than TF.

TF-binding scFv antibodies that do not compete with FVII in binding to TF are selected using sTF / FVIIa peptide hydrolysis assay. In this assay, TF-antibodies that compete with FVIIa for binding to TF are expected to inhibit the hydrolysis of chromogenic substrate S2266. 1 shows that the single-chain antibody scFv (TF) 3e10 did not inhibit FVIIa activity and actually increased that activity.

TF-binding scFv antibodies that inhibit FX activation are selected using PT assay and FX activation assay. In PT assays, TF-antibodies that extend PT are expected to inhibit FX activation. 3 shows that the single chain antibody scFv (TF) 3e10 extended PT, suggesting that the antibody inhibits FX activation. In FX activation assays, TF-antibodies that inhibit FXa activity are expected to inhibit the hydrolysis of chromogenic substrate S2222. 6 shows that the single chain antibody scFV (TF) 3e10 inhibited FXa activity.

TF-binding scFv antibodies that do not compete with FX in binding to TF are selected using FX activation assay. In this assay, TF-antibodies that do not compete with FX are expected to inhibit FX activation regardless of the concentration of FX used. 7 shows that the single chain antibody scFv (TF) 3e10 inhibited FXa activity in an FX concentration-dependent manner.

In a preferred embodiment of the invention, single chain antibodies (scFv (TF) 3e10) with a single V _H / V _L binding site for TF were identified. The amino acid sequence of scFv (TF) 3e10 is shown in Example 1 and corresponds to SEQ ID NO: 1. The DNA sequence encoding scFv (TF) 3e10 corresponds to SEQ ID NO: 2.

Analogs, fragments, derivatives and variants of the antibodies of the invention:

Analogs, fragments, derivatives or variants of the antibodies of the invention include (i) one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues) and the substituted amino acid residues are genetic code Coded or uncoded by; (ii) at least one amino acid residue comprises a substituent; (iii) the matured antibody is fused with another compound, such as a compound that increases the half-life of the antibody (eg, polyethylene glycol); Or (iv) an additional amino acid, such as a leader or secretory sequence, or a sequence used for purification of a mature antibody, may be fused with a mature antibody. Such analogs, fragments, derivatives and variants are deemed to be within the skill of those skilled in the art from the teachings herein.

Preferably, derivatives of the present invention will contain conservative amino acid substitutions (defined further below) at one or more expected residues, preferably non-essential amino acid residues. "Non-essential" amino acid residues are residues that can be altered from the wild-type protein sequence without altering the biological activity, and "essential" amino acids are residues necessary for biological activity. "Conservative amino acid substitutions" are those in which amino acid residues are replaced with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are defined in the art. These groups include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg , Glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (Eg threonine, valine, isoleucine) and amino acids having aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Non-conservative substitutions will be made to conservative amino acid residues or amino acid residues within a conservative protein domain only if such non-conservative substitutions are performed to select for antibody variants described further below. Fragments or biologically active moieties include polypeptide fragments suitable for use as medicinal and research reagents and the like. Such fragments include amino acid sequences sufficiently similar to the amino acid sequences of the antibodies of the invention, or amino acid sequences derived from amino acid sequences of said antibodies, which exhibit one or more of the activities of the polypeptides, but do not comprise the amino acid sequences of the full-length polypeptides disclosed herein. Small numbers of peptides are included.

Preferred derivatives of the invention also include mature antibodies fused with other compounds, such as compounds that increase the half-life of the polypeptide and / or reduce the potential immunogenicity of the polypeptide (eg, polyethylene glycol, "PEG"). do. PEG can be used to impart water solubility, size, relaxation of clearance through kidneys, and reduced immunogenicity (see, eg, US Pat. No. 6,214,966). In the case of PEGylation, fusion of the antibody with PEG can be accomplished by any means known to those skilled in the art. For example, PEGylation can be accomplished by first introducing cysteine mutations into the antibody followed by site-specific derivatization with PEG-maleimide. Cysteine can be added to the C-terminus of the peptide (see, eg, Tsutsumi et al. (2000) Proc. Natl. Acad. Sci. USA 97 (15): 8548-8553). Other modifications that can be made in the antibody include biotinylation. In certain cases, it may be useful for the antibody to be biotinylated to readily react with streptavidin. Methods for biotinylation of proteins are well known in the art. In addition, an N- or O-glycosylation site can be introduced into the antibody sequence so that post-translational N- or O-linked glycosylation of the antibody occurs in vivo.

Variants of the antibodies of the invention include polypeptides having an amino acid sequence sufficiently similar to that of the original antibody. The term “sufficiently similar” means a sufficient number of amino acid residues in which the first amino acid sequence is identical to the second amino acid sequence such that the first amino acid sequence and the second amino acid sequence have a common structural domain and / or common functional activity; Equivalent amino acid residues are meant to be contained in the minimum number. For example, amino acid sequences that contain a common structural domain and are at least about 45%, preferably about 75% to 98% identical, are defined herein as sufficiently similar. Preferably, the variant will be sufficiently similar to the amino acid sequence of the preferred antibody according to the invention. Variants include variants of antibodies encoded by polynucleotides of the invention or polynucleotides that hybridize under stringent conditions with their complements. Such variants generally retain the functional activity of the antibody according to the invention. Libraries of polynucleotide fragments can be used to generate a variety of fragment libraries for screening and later selection. For example, under conditions in which only one nick per molecule occurs, the double-stranded PCR fragment of polynucleotide is treated with nucleases, the double-stranded DNA is denatured, and the DNA is repaired to produce different nick products. Form double-stranded DNA, which may include sense / antisense pairs from, and treat with S1 nuclease to remove single-stranded DNA from the modified duplex, and write the resulting fragment library to the expression vector. Fragment libraries can be prepared by gating. By this method, expression libraries encoding N-terminal fragments and internal fragments of various sizes for the antibodies according to the invention can be derived.

Variants include antibodies that differ in amino acid sequence due to mutagenesis. For example, by performing mutagenesis according to recombinant DNA techniques well known in the art to modify the V _L and V _H domains of the Ig light and heavy chains, they have increased binding affinity for the FVIIa / TF complex compared to the free TF. Variants can be generated. Particularly preferred variants include antibodies with modified moieties within the complementarity determining regions of the V _L and V _H domains.

Variants also include antibodies that differ in amino acid sequence due to insertion or deletion of amino acid residues by mutagenesis. For example, mutagenesis may be performed using recombinant DNA techniques well known in the art to insert or delete amino acids at the N-terminal or C-terminal portions of the V _L or V _H domains of the Ig light and heavy chains. Variants that retain functional activity can be generated. Particularly preferred variants include single chain antibodies in which the amino acid residue of the V _L -V _H linker between the V _L and V _H domains is inserted or deleted. The V _L -V _H linker sequence of the single chain antibody shown in Example 1 is 5 amino acid residues in length. Other short linker sequences of 0-20 amino acids can be used and it will be evident that this antibody possesses substantially similar functional activity. Modifications of the V _L -V _H linkers present may be aimed at increasing the stability of the dimeric forms of single chain antibodies.

In one embodiment, the colorful variant library is generated by combinatorial mutagenesis at the nucleic acid level and encoded by the colorful gene library. The colorful variant libraries are, for example, such that a degenerate set of potential variant amino acid sequences can be expressed as individual polypeptides or alternatively as large fusion proteins (eg, for phage display) containing the set of sequences therein. Mixtures of synthetic oligonucleotides can be generated by ligation into gene sequences by enzymes. There are a variety of methods that can be used to generate a library of potential variants from the polyvalent oligonucleotide sequence. Chemical synthesis of the multigenic gene sequence can be performed in an automated DNA synthesizer, and then the synthetic gene can be ligated into a suitable expression vector. Using a multivariate gene set, one can provide all the sequences encoding a desired set of potential variant sequences in one mixture. Methods of synthesizing multivalent oligonucleotides are known in the art (eg, Narang (1983) Tetrahedron 39: 3, Itakura et al. (1984a) Annu. Rev. Biochem. 53: 323), (Itakura et al. (1984b) Science 198: 1056), Ike et al. (1983) Nucleic Acid Res. 11: 477).

Various techniques are known in the art for screening gene products of combinatorial libraries prepared by point mutations or truncation, and for screening cDNA libraries for gene products with selected properties. Such techniques can be adapted to rapidly screen gene libraries generated by combinatorial mutagenesis of antibodies for TF- or FVIIa / TF complex-binding activity or FX activation inhibitory activity. Typically, the most widely used techniques for performing high throughput assays to screen large gene libraries include cloning the gene library into replicable expression vectors, transforming the appropriate cells with the resulting vector library, and desired Expression of the combinatorial gene is included under conditions that facilitate the detection of the activity encoding the vector encoding the detected gene product. Recursive ensemble mutagenesis (REM), a technique for increasing the frequency of functional mutants within a library, can be used in conjunction with screening assays to identify desired variants.

Example 1 shows the amino acid sequence of one TF-binding single chain antibody, scFv (TF) 3e10 (SEQ ID NO: 1) and describes the V _L -V _H linker and the V _L and V _H domains. Variants having TF- or FVIIa / TF complex-binding activity or inhibiting FX activation may be mutated, e.g., of antibodies of the invention using the sTF / FVIIa peptide hydrolysis or FX activation assay described in Example 4. This can be confirmed by screening for combinatorial libraries of insertion, truncation or point mutants. Antibodies of the invention include antibodies of Examples 1 and 3 (SEQ ID NOs: 1 and 3), and antibodies with no substantial changes in the sequences derived therefrom. “Non-substantial change” includes any sequence, substitution or deletion variant that substantially maintains one or more biological functions of an antibody of the invention, eg, the ability to inhibit FX activation. Such functional equivalent thereof is preferably SEQ ID NO: 1 or a V _L or V _H region domain and at least about 90% identity of the single-chain antibody of SEQ ID NO: 3, and more preferably SEQ ID NO: 1 or SEQ ID NO: 3 single-chain antibody of the V _L or V _H region domains of the And at least 95% identity, even more preferably an antibody having at least 97% identity with a V _L or V _H region domain of a single chain antibody of SEQ ID NO: 1 or SEQ ID NO: 3, wherein said antibody has substantially the same biological activity It may also include part of. However, any antibody having a non-substantial change in amino acid sequence from the antibody of SEQ ID NO: 1 or SEQ ID NO: 3, demonstrating the functional equivalency described further herein, is included in the description of the present invention.

Pharmaceutical composition:

The present invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention may be prepared to administer an antibody of the invention having a desired degree of purity in combination with a pharmaceutically acceptable pharmaceutically effective amount of a carrier.

Antibodies of the invention can be used in pharmaceutical compositions for intravenous, subcutaneous or intradural administration. Thus, the antibodies described above are preferably combined with acceptable sterile pharmaceutical carriers such as 5% dextrose, lactate treated Ringer's solution, conventional saline, sterile water, or any other commercial physiological buffer designed for intravenous irrigation. Do. It will be appreciated that the choice of carrier solution, and the dosage and administration of the composition will vary depending upon the subject and the particular clinical environment, which is controlled by standard medical procedures.

In accordance with the methods of the invention, these pharmaceutical compositions may be administered in an amount effective to inhibit the pathological consequences associated with excessive thrombin production in the subject.

The antibody can be administered by intravenous bolus injection, constant intravenous irrigation, or a combination of the two routes. Alternatively or additionally, the antibody mixed with the appropriate excipient may be circulated from the intramuscular site. Systemic treatment with antibodies can be monitored by measuring the activated partial thromboplastin time (PT) in a series of blood samples taken from the patient. The clotting time observed in this assay is extended if the antibody is present in sufficient amounts in the circulation.

The recombinant antibodies and pharmaceutical compositions of the invention are useful for parenteral, topical, intravenous, oral or topical administration. Depending on the method of administration, the pharmaceutical composition can be administered in various unit dosage forms. For example, unit dosage forms can be administered in forms including, but not limited to, tablets, capsules, powders, solutions, emulsions, and the like.

Recombinant antibodies and pharmaceutical compositions of the invention are particularly useful for intravenous administration. Compositions for administration will typically comprise a solution of single chain antibody dissolved in a pharmaceutically acceptable carrier (preferably an aqueous carrier). For example, various aqueous carriers can be used, such as buffered saline and the like. These solutions are sterile and are generally free of unwanted substances. The composition can be sterilized by well known conventional sterilization techniques.

Pharmaceutical compositions for conventional intravenous administration can be readily determined by one skilled in the art. The amount administered is clearly specific to the protein and depends on its efficacy and pharmacokinetic profile. Actual methods of preparing compositions for parenteral administration are known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa (1980).

Compositions containing an antibody of the invention or a cocktail thereof (ie, a mixture with other proteins) can be administered as a therapeutic agent. In the field of treatment, the composition is administered to a patient suffering from a hemorrhagic disorder or disease in an amount sufficient to cure the bleeding or at least partially arrest the bleeding. A suitable amount to achieve this is defined as a "therapeutically effective amount." The amount effective for this use will depend on the severity of the disease and the general health of the patient.

The composition may be administered once or multiple times, depending on the dosage and frequency of administration required and acceptable by the patient. In any case, the composition must provide the protein of the invention in an amount sufficient to be effective for treating the patient.

Antibodies of the present invention or pharmaceutically acceptable compositions thereof may include the activity of the specific antibody employed; Metabolic stability and duration of action of the antibody; The age, body weight, general health, sex and diet of the patient; Mode of administration and time of administration; Excretion rate; Drug use; The severity of the specific disease state; And therapeutically effective amounts depending on a variety of factors including the host undergoing the therapy. In general, the therapeutically effective amount per day is from about 0.14 mg to about 14.3 mg of the antibody or pharmaceutically acceptable composition thereof per kg body weight per day, preferably from about 0.7 mg / kg body weight per day About 10 mg, most preferably about 1.4 mg to about 7.2 mg / kg body weight per day. For example, when administered to a person weighing 70 kg, the dosage range is from about 10 mg / day to about 1.0 g / day of the antibody or pharmaceutically acceptable composition thereof, preferably about 50 mg / day. Day to about 700 mg / day, most preferably about 100 mg / day to about 500 mg / day.

Cell and Gene Therapy:

In addition, the antibodies of the invention can also be used in accordance with the invention by expressing the antibody in vivo (often referred to as "gene therapy"). As such, for example, cells can be genetically engineered ex vivo with polynucleotides encoding the antibody (DNA or RNA), and then the engineered cells are provided to the patient to be treated with the antibody. Such methods are well known in the art. For example, retroviral particles containing RNA encoding the antibodies of the invention can be used to genetically engineer cells by procedures known in the art.

The antibodies of the present invention may also be used in accordance with the present invention by expressing the antibodies in vivo by a method called "gene therapy." As such, for example, the virus may be genetically engineered with a polynucleotide (DNA or RNA) encoding the antibody, followed by providing the engineered virus to a patient to be treated with the antibody. Such methods are well known in the art. For example, recombinant adenoviruses containing DNA encoding the antibodies of the invention can be genetically engineered by methods known in the art.

Local delivery of the anticoagulant antibodies of the invention using cell or gene therapy can provide therapeutic agents to the target region (endothelial cell connecting blood vessels).

Gene therapy of both in vitro and in vivo can be considered. Various methods of delivering a potential therapeutic gene to a defined cell population are known (see, eg, Muligan (1993) Science 260: 926-931). Such methods include the following delivery methods:

1) direct gene delivery (see, eg, Wolff et al. (1990) Science 247: 1465-1468);

2) DNA delivery mediated by liposomes (see, eg, Caplen et al. (1995) Nature Med. 3: 39-46, Crystal (1995) Nature Med. 1: 15-17), Gao and Huang (1991) Biochem. Biophys. Res. Comm. 179: 280-285);

3) DNA delivery mediated by retroviruses (see, eg, Kay et al. (1993) Science 262: 117-119, Anderson (1992) Science 256: 808-813);

4) DNA delivery mediated by DNA viruses (the DNA viruses include adenoviruses (preferably vectors based on Ad2 or Ad5), herpes viruses (preferably vectors based on herpes simplex virus) and parvoviruses (preferably Include "defective" or non- spontaneous parvovirus based vectors, more preferably vectors based on adeno-associated viruses, most preferably vectors based on AAV-2), for example, Ali et al. (1994) Gene Therapy 1: 367-384, US Patent No. 4,797,368, which is hereby incorporated by reference in its entirety, and US Patent No. 5,139,941, which is considered to be incorporated herein by reference. Reference).

The particular vector system for delivering the gene of interest will be chosen differently depending on various factors. One of the important factors is the nature of the target cell population. Although retroviral vectors have been widely studied and used in many gene therapy fields, they are generally not suitable for infecting non-dividing cells. Retroviruses also have the potential for tumorigenesis. However, recent developments in the field of lentiviral vectors can overcome some of these limitations (see Naldini et al. (1996) Science 272: 263-267).

Retroviruses from which the above-mentioned retroviral plasmid vectors may be derived include Moloney Murine leukemia virus, spleen necrosis virus, retroviruses such as Rous's sarcoma virus, Harvey Sarcoma virus, avian leukemia virus, gibbon monkey leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and breast tumor virus. In one embodiment, the retroviral plasmid vector is derived from Molonei murine leukemia virus.

Adenoviruses with the advantage of having a broad host range can infect stationary or finally differentiated cells, such as neurons or hepatocytes, and appear to be essentially free of tumors (eg, literature [ Ali et al. (1994), supra, p. 367). Adenovirus does not appear to integrate into the host genome. Because adenoviruses are external to the chromosome, the risk of mutagenesis by insertion is greatly reduced [Ali et al. (1994), supra, p. 373].

Adeno-associated viruses show similar advantages as vectors based on adenoviruses. However, AAV shows site-specific integration for human chromosome 19 [Ali et al. (1994), supra, p. 377].

In a preferred embodiment, the DNA encoding the antibody of the invention is a cell or gene for a disease, including but not limited to deep vein thrombosis, disseminated intravascular coagulation, acute heart syndrome, or cancer with signs of coagulation Used in therapy.

According to this embodiment, the cell or gene therapy with the DNA encoding the antibody of the invention is provided to the patient in need thereof concurrently with or shortly after diagnosis.

Those skilled in the art will appreciate that any suitable gene therapy vector containing DNA encoding the antibodies of the invention or DNA encoding analogs, fragments, derivatives or variants of the antibodies of the invention may be used according to the above embodiments. Techniques for constructing such vectors are known (see, eg, Anderson, W. F. (1998) Nature 392: 25-30; Verma I. M. and Somia, N. (1998) Nature 389: 239-242). Introduction of the antibody-DNA containing vector to the target site can be accomplished using known techniques.

The cell or gene therapy vector comprises one or more promoters. Suitable promoters that can be used include those described by Miller et al. (1989) Biotechniques 7 (9): 980-990, retrovirus LTR; SV40 promoter; And human cytomegalovirus (CMV) promoters, or any other promoters (such as but not limited to cellular promoters such as eukaryotic cell promoters, including but not limited to histone, pol III and β-actin promoters). It doesn't work. Other viral promoters that can be used include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of suitable promoters will be apparent to those skilled in the art from the teachings contained herein.

Nucleic acid sequences encoding the antibodies of the invention are under the control of suitable promoters. Suitable promoters that can be used include adenovirus promoters, for example adenovirus major late promoters; Or heterologous promoters such as the cytomegalovirus (CMV) promoter; Respiratory syncytial virus (RSV) promoter; Inducible promoters such as the MMT promoter, metallothionein promoter; Heat shock promoters; Albumin promoter; ApoAI promoter; Human globin promoter; Viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; Retrovirus LTR (including the modified retrovirus LTR described above); β-actin promoter; And human growth hormone promoters.

Producer cell lines are formed by transducing packaging cell lines using retroviral plasmid vectors. Examples of packaging cells that can be transfected include PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X; VT-19-17-H2, ψCRE, ψCRIP, GP + # 86, GP + envAm12, and DAN cell lines (Miller (1990) Human Gene Therapy 1: 5-14, which is incorporated herein by reference in its entirety) Described), but is not limited thereto. The vector may transduce the packaging cells by any means known in the art. Such methods include, but are not limited to, electroporation, the use of liposomes and CaP0 ₄ precipitation. As an alternative, the retroviral plasmid vector can be encapsulated in liposomes or linked to a lipid and then administered to a host. Producer cell lines produce infectious retroviral vector particles comprising nucleic acid sequence (s) encoding a polypeptide. The retroviral vector particles can then be used to transduce eukaryotic cells in vitro or in vivo. Transduced eukaryotic cells will express the nucleic acid sequence (s) encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

Another method for gene therapy is "transkaryotic therapy," where cells of the patient are treated ex vivo to induce resting chromosomal genes to produce the desired protein after reintroduction into the patient. Transkeriotic therapies assume that the individual has the normal complement of the genes required for activation. Transkeriotic therapy involves introducing a promoter or other exogenous regulatory sequence capable of activating nascent genes into the chromosomal DNA of a patient's cells in vitro, culturing and selecting active protein-producing cells, and then Reintroducing the activated cells to the patient so that these cells are fully established. The “gene activated” cells then produce the desired protein for a somewhat significant time, perhaps for the life of the patient. US Pat. Nos. 5,641,670 and 5,733,761, which are incorporated by reference in their entirety, disclose this concept in detail.

Kit:

The invention also relates to kits for research or diagnostic purposes. Typically, the kit includes one or more containers containing the antibodies of the invention. In a preferred embodiment, the kit comprises a container containing a single chain antibody in a form suitable for derivatization with a second molecule. In a more preferred embodiment, the kit comprises a container containing the antibody of SEQ ID NO: 1 or SEQ ID NO: 3.

In other embodiments, the kit may contain a DNA sequence encoding an antibody of the invention. Preferably, the DNA sequences encoding such antibodies are provided in plasmids suitable for transfection and expression by host cells. The plasmid may contain a promoter (often an inducible promoter) that regulates expression of DNA in host cells. The plasmid may also contain appropriate restriction sites that facilitate insertion of other DNA sequences into the plasmid to prepare a variety of antibodies. The plasmid may also contain a number of other elements that facilitate cloning and expression of the encoded protein. Such elements are well known to those skilled in the art and include, for example, selectable markers, initiation codons and termination codons, and the like. In a more preferred embodiment, the kit comprises a container containing the DNA sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

Treatment Indications:

Diseases in which thrombus formation plays a pathologically significant role include myocardial infarction, disseminated intravascular coagulation, deep vein thrombosis, pulmonary embolism, ischemic attack, septic shock, acute respiratory disorder syndrome, unstable angina and other arteries and Venous obstruction. Antibodies of the invention are useful in all of these diseases and in other diseases in which thrombus formation is a pathological cause. Other pathological conditions in which the antibodies of the invention may be useful include cancers with coagulation and inflammatory symptoms. In addition, antibodies may be useful in skin and vein transplants and organ transplants. Useful means that the antibody is useful for treatment to prevent disease or to prevent the disease from developing into more serious situations. In addition, the antibodies of the invention also provide safe and effective anticoagulants in patients receiving bioartificial inserts, such as, for example, heart valves. Such antibodies may replace heparin and warfarin, for example in the treatment of pulmonary embolism or acute myocardial infarction. Antibodies of the invention can also be used to coat medical devices that are subject to coagulation.

analysis:

Numerous experimental assays can be used to determine the in vitro anticoagulant activity of the antibodies of the invention. Anticoagulant effects of antibodies can be determined using plasma coagulation time assays, eg, activated partial thromboplastin time (“APTT”), thrombin coagulation time (“TCT”), and / or prothrombin time (“PT”). It can be measured. These assays are distinguished from the different mechanisms of coagulation inhibition and include the activation of protein C. The prolongation of clotting time in either of these assays demonstrates that the molecule can inhibit clotting in plasma.

Using this assay, antibodies with anticoagulant activity capable of binding to TF in both the purified system and the plasma environment are identified. Thereafter, other assays are used to inhibit the formation of fibrin from fibrinogen by other activities of the antibodies of the invention, such as thrombin catalysis [Jakubowski, H. V. et al. (1986) J. Biol. Chem. 261 (8): 3876-3882], inhibition of thrombin activation of factor V [Esmon, C. T. et al. (1982). J. Biol. Chem. 257 (14): 7944-7947], promoting inhibition of thrombin by antithrombin III and heparin cofactor II [Esmon, N. L. et al. (1983) J. Biol. Chem. 258 (20): 12238-12242], inhibition of thrombin activation of factor XIII [Polgar, J. et al. (1987) Thromb. Haemost. 58 (1): 140], inhibition of thrombin mediated inactivation of protein S [Thompson, E. A. and Salem, H. H. (1986) J. Clin. Inv. 78 (1): 13-17], and inhibition of thrombin mediated platelet activation and aggregation [Esmon, N. L. et al. (1983), supra].

In vitro potency or in vitro binding affinity of the antibodies of the invention were determined using the following assays detailed in Example 4: 1) sTF / FVIIa peptide hydrolysis assay; 2) factor X activation assay; 3) PT analysis; And 4) microcalorimetric analysis.

In carrying out the methods of the present invention, of course, it is not intended to be limiting to particular buffers, media, reagents, cells and culture conditions, but to include all relevant materials which are recognized by the person skilled in the art as being intended or useful in the particular situation in question. It should be understood as being interpreted. For example, often replacing the buffer system or culture medium with another one can still achieve similar results if not identical. Those skilled in the art will have sufficient knowledge of the above systems and methods so that such substitutions can be made without undue experimentation to optimally serve a given purpose in using the methods and procedures disclosed herein.

The invention will be further described by the following non-limiting examples as a guide to assist in the practice of the invention. In applying the disclosure of the examples, it should be clear that other different embodiments of the methods disclosed in accordance with the present invention will be apparent to those skilled in the art.

In the above disclosure and the following examples, all temperatures are described in uncorrected degrees Celsius, and all parts and ratios are by weight unless otherwise indicated.

The entire disclosures of all applications, patents, and publications cited above are hereby incorporated by reference.

*****

Example 1

Single-chain anti-TF antibody construct scFv (TF) 3e10

The single chain anti-TF antibody scFv (TF) 3e10 (SEQ ID NO: 1) is composed of a signal peptide (-18 to -1), a V _H domain (1 to 126), a V _H -V _L linker (127 to 131), a V _L domain ( 132-246, and e-tag sequences 247-264. The scFv (TF) 3e10 DNA sequence (SEQ ID NO: 2) encodes the amino acid sequence of SEQ ID NO: 1.

Example 2

Single chain anti-TF antibody construct scFv (TF) 3e10Δ

The single chain anti-TF antibody scFv (TF) 3e10Δ (SEQ ID NO: 3) is a signal peptide (-18 to -1), V _H domain (1 to 126), V _H -V _L linker (127 to 131), and V _L It consists of domains 132-243. scFv (TF) 3e10Δ is different due to the deletion of three amino acids (GAP) at the N-terminus of the scFv (TF) 3e10 and the V _L domain and the C-terminal e-tag sequence. The scFv (TF) 3e10Δ DNA sequence (SEQ ID NO: 4) encodes the amino acid sequence of SEQ ID NO: 3.

Example 3

Expression of anti-TF antibodies in bacteria and mammalian cells

As described above, the six different single-chain antibodies, scFv (TF) 2c1, scFv (TF) 2c11, scFv (TF) 2d3, scFv (TF) 2h6, scFv (TF) 3e10 and scFv (TF) 3h2, were TF- Identification from binding phage; Overexpressed in E. coli and affinity purified using an e-tag affinity column. The affinity of the six purified antibodies to sTF was measured using BIAcore, and then these antibodies were characterized by hydrolysis, FX activation, PT and microcalorimetry assays as described in Example 4. And the results are described in Example 5.

scFv (TF) 3e10 antibody (SEQ ID NO: 1) was also expressed in CHO cells. The expression plasmid contained both a DNA sequence encoding scFv (TF) 3e10 (SEQ ID NO: 2), hygromycin B and a DHFR selection marker. Original selection was performed at 400 μg / ml of hygromycin to select populations. The resulting populations were then subjected to 100 nM methotrexate selection. During this screening, cells amplified copies of the DNA region and target gene containing the selection marker were selected from the population. As a result of this selection, the expression level increased from about 0.3 mg / L to about 6 mg / L.

Example 4

In vitro potency and binding affinity analysis

1. sTF / FVIIa Peptide Hydrolysis Assay

The principle of this analysis is described below. The tripeptide p-nitroanilide amide bond of the substrate is hydrolyzed by the sTF / FVIIa complex. The free chromophore product, p-nitroanilide, is monitored at 405 nm and the molar extinction coefficient of 9920 M ⁻¹ cm ⁻¹ is used to calculate the concentration of product formed per unit time. The initial ratio was put into the four parameter equation to determine the IC ₅₀ value (C): Y = (AD) / (1+ (x / C) ^ B) + D

H-D-Val-Leu-Arg-p-NA ⇒ H-D-Val-Leu-Arg + p-NA

S2266 Substrate Tripeptide Chromophore                 

Reagents and Solutions:

Assay buffer: 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl ₂ , 0.1% BSA, pH 7.5

2. Human FVIIa (HCVIIA-0060, Haematologic Technologies Inc.): 10 × Working Solution—Prepare a 20 nM solution in assay buffer before use.

3. Soluble TF (Berlex): 10 × Working Solution—Prepare 30 nM solution in assay buffer before use.

4. Coloring Substrate S2266 (Kabi Pharmacia Hepar Inc.): Stock: 10 mM in H ₂ O, stored at 4 ° C. 2.5 × Working Solution—Prepare a 2.5 mM solution in assay buffer before use.

5. Antibodies: Prepare 2.5 × dilutions in assay buffer prior to use.

Analysis condition:

Assays were performed at room temperature in 96-well microtiter plates. Final concentrations of the components were as follows:

sTF 3 nM

Varies from 1000 to 0.625 nM antibody

FVIIa 2 nM

S2266 1 mM                 

Analytical Procedure:

1. Pipette 0.1 ml of 2.5 × AB (or buffer control) into each well.

2. Add 0.025 ml of 10 × sTF and incubate for 10 minutes at room temperature with slight shaking.

3. Add 0.025 ml of 10 × FVIIa and incubate for 10 minutes at room temperature with slight shaking.

4. Add 0.1 ml of 2.5 × S2266 substrate and immediately transfer the plate into the plate reader and measure the enzyme reaction rate for 15 minutes at 405 nm at 10 second intervals.

This assay can be used to determine the apparent K _D value of the binding of the antibody of the invention to the sTF or FVIIa / TF complex and determine whether the antibody of the invention competes with FVII for binding to TF.

2. Factor X Activation Assay

The principle of this analysis is described below. FVIIa is incubated with recombinant human TF vesicles to form a protease complex capable of activating the substrate FX. This complex is formed in the presence (or absence) of antibodies of different concentrations, and then the substrate FX is introduced and the reaction proceeds to form the product, active protease FXa, wherein the active protease FXa is the p-nitroanilide of the chromogenic substrate S2222. Amide bonds can be hydrolyzed. The free chromophore product, p-nitroanilide, is monitored at 405 nm and the molar extinction coefficient of 9920 M ⁻¹ cm ⁻¹ is used to calculate the concentration of product formed per unit time. The initial ratio was put into the four parameter equation to determine the IC ₅₀ value (C):

Y = (A-D) / (1+ (x / C) ^ B) + D

Bz-Ile-Glu-Gly-Arg-p-NA ⇒ Bz-Ile-Glu-Gly-Arg-OH + p-NA

S2222 Substrate Chromophore

Reagents and Solutions:

Assay buffer: 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl ₂ , 0.1% BSA, pH 7.5

2. Human FVIIa (HCVIIA-0031, Hemertologic Technologies Inc.): 4 × Working Solution—Prepare a 100 pM solution in assay buffer before use.

3. Recombinant human TF (reconstructed in our laboratory from Innovin, Dade): Working solution-Prepare 1: 480 dilution in assay buffer prior to use.

4. Human Factor X (HCX-0060, Hemertologic Technologies Inc.): 4 × Working Solution—Prepare 1000 nM solution in assay buffer before use.

5. Coloring Substrate S2222 (Kabi Pharmacia Hepar Inc.):

Stock: 6 mM in H ₂ O, stored at 4 ° C.

Working solution—Prepare a 0.78 mM solution in 3.57 mM EDTA (to stop the reaction), 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 before use.

6. Antibodies:

Prepare 4 × dilution in assay buffer before use.

Analysis condition:

Assays were performed at room temperature in 96-well microtiter plates. Final concentrations of the components were as follows:

1/4 of rTF vesicle 1: 480 dilution

Varies from 1000 to 0.625 nM antibody

FVIIa 25 pM

FX 250 nM

S2222 0.546 mM

Analytical Procedure:

1. Pipette 0.015 ml of 4 × AB (or buffer control) into each well.

2. Add 0.015 ml of 4 × rTF vesicles.

3. Add 0.015 ml of 4 × FVIIa and incubate for 10 minutes at room temperature with slight shaking.

4. Add 0.015 ml of 4 × FX and incubate at room temperature for 5 minutes with slight shaking.

5. Add 0.14 ml of S2222 substrate, immediately transfer plate into plate reader and measure enzyme reaction for 15 minutes at 405 nm at 10 second intervals.

This assay can be used to determine if the antibodies of the invention activate FX by inhibiting the FVIIa / TF complex and whether the antibodies of the invention compete with FX for binding to the FVIIa / TF complex.

3. Prothrombin Time (PT) Analysis

For a standard PT reaction, an appropriate concentration of 90 μl of antibody or PBS is added to 20 μl of thromboplastin IS (Dade) and 90 μl of 25 mM CaCl ₂ in cuvette. The mixture was incubated at 37 ° C. for 1 minute, then 100 μl of citrated plasma (Helena Laboratories) was added. Alternatively, an appropriate volume of concentrated antibody was added to 100 μl of recombinant human thromboplastin and approximately 2 minutes later, 100 μl of reconstituted human plasma was added. The coagulation time for each individual coagulation assay was determined by taking the average of the two measurements using an Electra 900C Coagulometer (Hemoliance) and the mean value determined from replicate analysis (n = 3 or 4). . Dose response graphs were generated for each inhibitor, and regression analysis was used to calculate the concentration (in nM units) needed to double the clotting time.

The assay can be used to assess the effect of the antibodies of the invention on external blood coagulation pathways. The amount of antibody needed to double PT can be determined and compared to other anticoagulants to assess the relative efficacy of the antibodies of the invention.

4. Micro Calorimetry Analysis

The binding affinity (K _D ) of the antibodies of the invention to TF or FVIIa / TF complexes alone was determined using microcalorimetry (isothermal titration calorimetry) assay. This assay was performed using a MicroCal VP-ITC instrument. A 2.3-fold molar excess of FVIIai was added to sTF to form the FVIIa / TF complex. Size exclusion chromatography was used to confirm that the sTF in this assay formed a complete complex. To determine antibody affinity for the complex, 1.2 μM VIIa / TF complex was added to the microcalorimeter cell and 65 μM antibody was placed in a syringe. To determine antibody affinity for sTF alone, 10 μM sTF was placed in the cell and 141 μM antibody was placed in the syringe. Data analysis was performed using MicroCal Origin software. Data was fitted to a single binding site.

Example 5

In vitro Features of Anti-TF Antibodies of the Invention

Six different TF-binding antibodies were isolated from a complete human single chain antibody phage display library: scFv (TF) 2c1, scFv (TF) 2c11, scFv (TF) 2d3, scFv (TF) 2h6, scFv (TF) 3e10 and scFv (TF) 3h2. This was measured using BIAcore. The affinity of the sTF-binding antibodies expressed in E. coli was 35 to 470 nM. The sTF / VIIa peptide hydrolysis assay described in Example 4 was used to determine if the antibody blocked the formation of the active VIIa / TF complex. In this assay, the binding of VIIa and sTF accelerated the cleavage rate to the chromogenic peptide substrate S2266 by more than 20 times. Antibodies that inhibit the binding of FVIIa to TF block this acceleration. Five of the single-chain antibodies, scFv (TF) 2c1, scFv (TF) 2c11, scFv (TF) 2d3, scFv (TF) 2h6 and scFv (TF) 3h2, inhibited S2266 hydrolysis, which prevented the antibodies from FVIIa and sTF Suggests inhibiting binding of (Fig. 1). In contrast, the single-chain antibody scFv (TF) 3e10 did not inhibit the sTF / VIIa peptide hydrolysis assay, which in fact increased the rate of hydrolysis of S2266, indicating that scFv (TF) 3e10 was associated with FVIIa. Suggests increasing the affinity of sTF for sTF. Using the sTF / VIIa peptide hydrolysis assay, scFv (TF) 3e10 antibody increased the affinity of FVIIa for sTF and reduced K _{D (apparent)} by 5 fold (FIG. 2). scFv (TF) 3e10 did not affect the rate of hydrolysis by FVIIa in the absence of sTF, suggesting that the antibody does not interact with FVIIa alone. K _D of the scFv (TF) 3e10 antibody against sTF measured using sTF / FVIIa peptide hydrolysis assay was 65.4 nM (FIG. 4). Microcalorimetry assays were used to compare the affinity of scFv (TF) 3e10 for TF with the FVIIa / TF complex. This experiment revealed that scFv (TF) 3e10 expressed by mammalian cells had about 20-fold higher affinity for the TF / FVIIa complex compared to free sTF (33 nM vs 600 nM, FIG. 5).

Six TF-binding single chain antibodies expressed in bacteria were compared using FX activation assay and PT assay. None of the five antibodies scFv (TF) 2c1, scFv (TF) 2c11, scFv (TF) 2d3, scFv (TF) 2h6 and scFv (TF) 3h2, which inhibited the hydrolysis rate of S2266 by the sTF / FVIIa complex PT was not extended beyond the PBS buffer control (FIG. 3). In contrast, scFv (TF) 3e10 antibody did not have the highest affinity as measured by BIAcore and increased the affinity of FVIIa for sTF, but scFv (TF) 3e10 inhibited FX activation in the group (FIG. 6, data not shown) was the only antibody with prolonged clotting time in the PT assay (FIG. 3). The IC ₅₀ of the scFv (TF) 3e10 (dimer) antibody on inhibition in the FX activation assay was 0.44 nM (FIG. 6). Finally, FX activation assays were used to determine whether the scFv (TF) 3e10 antibody competes with FX for binding to the FVIIa / TF complex. scFv (TF) 3e10 dose-dependently inhibited FX activation at the same K _{D (app)} at all concentrations of substrate FX, and these results indicate that scFv (TF) 3e10 does not compete with FX and does not compete with TF or at the same site as FX. Imply that it does not bind to the FVIIa / TF complex (FIG. 7).

ScFv (TF) 3e10 antibodies were identified based on binding to recombinant human soluble TF. Sequence homology of TF between human and murine or human and rabbit is 58% and 71%, respectively. The antibody appears to bind to specific epitopes on human TF that interfere with the activation of FX by the FVIIa / TF complex. Physiologically, such antibodies have advantages over antibodies that compete with FVII or FVIIa that bind TF. The K _D of FVIIa to soluble TF is about 10 nM (FIG. 2), which is the combination of FVIIa and sTF (4.8 nM, Neuenschwander, PF and Morrissey, JH (1994) J. Biol. Chem. 269 (11) : 8007-8013) or published values for binding of FVII, FVIIa and DIP inactivation-FVIIai with reconstituted full-length TF in neutral phosphatidylcholine vesicles (Bach R. et al. (1986) Biochemistry 25: 4007-4020) Matches The affinity of FVII or FVIIa for binding to full-length TF is due to the interaction of the GLA domain of FVII or FVIIa with the charged membrane surface [Neuenschwander, PF and Morrissey, JH (1994) supra], whereby charged phosphatidylserine is incorporated into phospholipid vesicles. Greatly increased when included [Bach R. et al. (1986) supra]. Under these optimal conditions, FVIIa binds with full length TF with very high affinity (41 pM). TF antibodies that compete with FVII or FVIIa binding will have difficulty competing against these high affinity FVIIa / TF complexes. In contrast, scFv (TF) 3e10 antibodies not only have greater affinity for the FVIIa / TF complex than TF, but also do not compete with FVIIa for binding to TF. Antibodies such as scFv (TF) 3e10 that do not compete with FVIIa will inhibit the activation of FX regardless of the plasma concentration of FVII which is about 10 nM.

In addition, scFv (TF) 3e10 antibodies have an advantage over antibodies that compete with FX for binding to TF. The K _m of FX for the VIIa / TF complex is 0.061 to 0.099 μM based on the data shown in FIG. 7 and is a published value (0.1 μM, Baugh, RJ et al. ): 28826-28833). The concentration of FX in human plasma is 140 nM (1.4-2 fold K _m ). Antibodies such as scFv (TF) 3e10 do not compete with FX as shown in FIG. 7 and will inhibit the activation of FX regardless of the plasma concentration of FX.

Example 6

In Vivo Rat Thromboembolism Model

The TF-binding single chain antibody scFv (TF) 3e10 is specific for primate TF. Development of a model of thromboembolism induced by human TF (thromboplastin preparation containing human recombinant TF, Ortho) in conscious male Sprague-Dawley rats (350-400 g, n> 7 / group) It was. In this model of disseminated intravascular coagulation (DIC), thromboplastin injection led to TF inducing pulmonary fibrin deposition, respiratory failure, and death. A predetermined dose of scFv (TF) 3e10 or vehicle expressed by mammalian cells was injected into the tail vein and 15 minutes later thromboplastin (0.5 ml / kg) was injected by bolus injection. In the vehicle treated group, this dose of TF typically exhibited 60% mortality (LD ₆₀ ) within 5 minutes after thromboplastin injection. Rats were scored according to the following morbidity-mortality scoring system: 0 = no effect; 1 = slight shortness of breath (recovered within 30 minutes); 2 = severe shortness of breath (frequent recovery, requiring more than 60 minutes); And 3 = death. Mean scores were used to compare the efficacy of different treatment groups. The result of using this in vivo assay is shown in FIG. 8. Antibodies of the invention were able to alleviate death and respiratory disorders in this assay.

The above examples could be repeated to have similar results by replacing generally or specifically described reactants and / or by carrying out the conditions of the present invention for those used in the above examples.                 

Although the present invention has been described with respect to the production of specific antibody constructs, it is evident that the variations and alternatives of the present invention can be performed without departing from the spirit and scope of the present invention.

<110> Light, David McLean, Kirk <120> Novel Tissue Factor Targeted Antibodies as Anticoagulants <130> 52295AWOM2 <150> US 60 / 376,566 <151> 2002-05-01 <160> 4 <170> PatentIn version 3.1 <210> 1 <211> 282 <212> PRT <213> Artificial Sequence <220> Amino acid sequence of scFv (TF) 3e10 antibody <400> 1 Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Val 1 5 10 15 Phe Pro Glu Met Ala Gln Val Asn Leu Arg Glu Ser Gly Gly Thr Leu             20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe         35 40 45 Ser Phe Thr Asp Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys     50 55 60 Glu Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Gly Ser Thr Tyr 65 70 75 80 Tyr Ala Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser                 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr             100 105 110 Ala Val Tyr Tyr Cys Ala Arg Val Leu Ser Leu Thr Asp Tyr Tyr Trp         115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala     130 135 140 Gly Gly Gly Gly Ser Gly Ala Pro Asn Phe Met Leu Thr Gln Pro His 145 150 155 160 Ser Val Ser Ala Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Thr Arg                 165 170 175 Ser Ser Gly Ser Val Ala Ser Tyr Tyr Val Gln Trp Tyr Gln Gln Arg             180 185 190 Pro Gly Ser Ser Pro Thr Thr Val Ile Tyr Glu Asp Asn His Arg Pro         195 200 205 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Thr Ser Ser Asn     210 215 220 Ser Ala Ser Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp Glu Ala Asp 225 230 235 240 Tyr Tyr Cys Gln Ser Tyr Asp Ser Asn Asn Leu Val Val Phe Gly Gly                 245 250 255 Gly Thr Lys Leu Thr Val Leu Gly Ala Ala Ala Gly Ala Pro Val Pro             260 265 270 Tyr Pro Asp Pro Leu Glu Pro Arg Ala Ala         275 280 <210> 2 <211> 849 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding scFv (TF) 3e10 antibody <400> 2 atgcttgggg tcctggtcct tggcgcgctg gccctggcag gcctggtctt ccccgagatg 60 gcccaggtca acttaaggga gtctggggga accttggtcc agcctggggg gtccctgaga 120 ctctcctgtg cagcctctgg attcagtttc actgacgcct ggatgagctg ggtccgccag 180 gctccaggga aggagctgga gtgggtctca agtattagtg gtagtggtgg aagcacatac 240 tacgcaggct ccgtgaaggg ccggttcacc atctccagag acaattccaa gaacacgctg 300 tatctgcaaa tgaacagcct gagagccgag gacacggccg tatattactg tgcgagagta 360 ttatcgctga ccgattacta ctggtacggc atggacgtct ggggccaagg caccctggtc 420 accgtctcgg ccggtggcgg cggatctggc gcgccaaatt ttatgctgac tcagccccac 480 tctgtgtcgg cgtctccggg gaagacggta accatctcct gcacccgcag cagtggcagc 540 gttgccagct actatgtgca gtggtaccag cagcgcccgg gcagttcccc caccactgtg 600 atctatgagg ataaccacag accctctggg gtccctgatc ggttctctgg ctccatcgac 660 acctcctcca actctgcctc cctcaccatc tctggactga agactgagga cgaggctgac 720 tactactgtc agtcttatga tagcaacaac cttgtggttt tcggcggagg gaccaagctg 780 accgtcctag gtgcggccgc aggagctccg gtgccggatc cggatccgct ggaaccgcgt 840 gccgcatga 849 <210> 3 <211> 261 <212> PRT <213> Artificial Sequence <220> Amino acid sequence of scFv (TF) 3e10delta antibody <400> 3 Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Val 1 5 10 15 Phe Pro Glu Met Ala Gln Val Asn Leu Arg Glu Ser Gly Gly Thr Leu             20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe         35 40 45 Ser Phe Thr Asp Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys     50 55 60 Glu Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Gly Ser Thr Tyr 65 70 75 80 Tyr Ala Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser                 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr             100 105 110 Ala Val Tyr Tyr Cys Ala Arg Val Leu Ser Leu Thr Asp Tyr Tyr Trp         115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala     130 135 140 Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro His Ser Val Ser 145 150 155 160 Ala Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly                 165 170 175 Ser Val Ala Ser Tyr Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser             180 185 190 Ser Pro Thr Thr Val Ile Tyr Glu Asp Asn His Arg Pro Ser Gly Val         195 200 205 Pro Asp Arg Phe Ser Gly Ser Ile Asp Thr Ser Ser Asn Ser Ala Ser     210 215 220 Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys 225 230 235 240 Gln Ser Tyr Asp Ser Asn Asn Leu Val Val Phe Gly Gly Gly Thr Lys                 245 250 255 Leu Thr Val Leu Gly             260 <210> 4 <211> 783 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding scFv (TF) 3e10delta antibody <400> 4 atgcttgggg tcctggtcct tggcgcgctg gccctggcag gcctggtctt ccccgagatg 60 gcccaggtca acttaaggga gtctggggga accttggtcc agcctggggg gtccctgaga 120 ctctcctgtg cagcctctgg attcagtttc actgacgcct ggatgagctg ggtccgccag 180 gctccaggga aggagctgga gtgggtctca agtattagtg gtagtggtgg aagcacatac 240 tacgcaggct ccgtgaaggg ccggttcacc atctccagag acaattccaa gaacacgctg 300 tatctgcaaa tgaacagcct gagagccgag gacacggccg tatattactg tgcgagagta 360 ttatcgctga ccgattacta ctggtacggc atggacgtct ggggccaagg caccctggtc 420 accgtctcgg ccggtggcgg cggatctaat tttatgctga ctcagcccca ctctgtgtcg 480 gcgtctccgg ggaagacggt aaccatctcc tgcacccgca gcagtggcag cgttgccagc 540 tactatgtgc agtggtacca gcagcgcccg ggcagttccc ccaccactgt gatctatgag 600 gataaccaca gaccctctgg ggtccctgat cggttctctg gctccatcga cacctcctcc 660 aactctgcct ccctcaccat ctctggactg aagactgagg acgaggctga ctactactgt 720 cagtcttatg atagcaacaa ccttgtggtt ttcggcggag ggaccaagct gaccgtccta 780 ggt 783  

Claims (25)

Comprising the amino acid sequence of SEQ ID NO: 1 or 3, An anticoagulant antibody that binds with greater affinity for Factor VIIa / Tissue Factor (FVIIa / TF) complexes than Tissue Factor (TF) alone. The antibody of claim 1, wherein the antibody binds at least twice as much affinity for the FVIIa / TF complex as measured by microcalorimetric analysis than TF alone. The antibody of claim 2, which binds with affinity of at least five times greater than the FVIIa / TF complex than the TF alone. The antibody of claim 3, wherein the antibody binds at least 10-fold greater affinity for the FVIIa / TF complex than the TF alone. The antibody of claim 1 which is a monoclonal antibody. The antibody of claim 5, which is a single chain antibody, Fab dimer antibody or IgG antibody. The antibody of claim 6 which is a single chain antibody. 8. The antibody of claim 7, wherein the antibody does not compete with at least one coagulation factor selected from the group consisting of Factor VII (FVII), Factor IX (FIX), and Factor X (FX) in binding to TF. The antibody of claim 8, wherein said coagulation factors are FVII and FX. The antibody of claim 1, wherein the glycosylated antibody. The antibody of claim 1, wherein the antibody is modified by the addition of polyethylene glycol. The antibody of claim 1, wherein the antibody is biotinylated for binding to streptavidin. delete A pharmaceutical composition for preventing thrombus formation, comprising a therapeutically effective amount of the antibody of claim 1 and a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 14 for preventing ischemic attack, thrombotic complications after angioplasty or microvascular surgery in microvascular surgery. Relieve coagulation in deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), acute heart syndrome, or advanced cancer in a patient, comprising a therapeutically effective amount of the antibody of claim 1 and a pharmaceutically acceptable excipient Pharmaceutical composition for treatment. A pharmaceutical composition for modulating an inflammatory response selected from the group consisting of sepsis, skin and vein transplantation, and organ transplantation, comprising a therapeutically effective amount of the antibody of claim 1 and a pharmaceutically acceptable excipient. delete The antibody of claim 1, which can be used to form a coating that prevents thrombus formation on the surface of a medical device in contact with blood. A kit comprising the antibody of claim 1. Kit comprising a DNA sequence encoding the antibody of claim 1. delete Comprises the amino acid sequence of SEQ ID NO: 1 or 3, An anticoagulant antibody that binds with greater affinity to the FVIIa / TF complex than TF alone, and does not compete with FVII and FX in binding to TF. delete A polynucleotide sequence encoding the antibody of claim 23 comprising the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

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